DESCRIPTION: (Applicant's Abstract) This investigation will study modified recombinant Baculovirus (rBV) as a gene vector in ocular tissues in vitro and in vivo. Baculovirus, an insect virus, is able to transfer large amounts of genetic material into target cells. Preliminary data suggests that the rBV is an efficient gene vector in cultured ocular and non-ocular cells as well as in certain ocular tissues in vivo, such as the retinal pigment epithelium (RPE) and corneal endotheflum. Gene transfer to ocular cells in vitro and in vivo is critical to further the understanding of ocular gene structure and function and potentially as a gene therapy vector. The specific aims of this project are 1) To engineer and evaluate rBV as a gene vector for mammalian cells in vitro for both long and short-term transgene expression; 2) To evaluate rBV vectors ability to transfer exogenous genes in the murine retina and other ocular tissues in vivo; and 3) To optimize production, purificat.ion and in vivo delivery of rBV vectors to the murine retina. To achieve these aims, commercially available rBV bacmids will be modifled for mammalian expression and be tested in vitro on RPE cells and HEK cells. Once the rBV synthesis is verified and expression is achieved in vitro, the same vector will be used to transfer marker genes to mouse retinas and other ocular tissue. A vector containing an RPE65 gene will be synthesized and' used to attempt to rescue the retinal degeneration in the RPE65 -/transgenic mouse model. Lastly, increasing the purity and titers of rBV, suppressing the immune system in mice prior to transfection and by adding additional genetic material to promote long-term expression, will optimize the vector system. If successful, the rBV system will prove to be a valuable tool for studying the structure and function of the normal and diseased retina and other ocular tissues in vitro and in vivo. With additional optimization, rBV may be an effective new gene therapy vector to treat retinal and other ocular diseases.